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        <title>Primer Design</title>
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        <h1 align="center">Primer Design</h1>
		<p>Although primer design is often characterized as a complex process 
		best relegated to sophisticated computer programs, in practice primer 
		performance has been found to be more dependent on assay design than 
		many of the esoteric parameters used by primer design programs. This is 
		not to say that primer-specific parameters are unimportant, 
		but rather that other factors, such as the enzyme formulation and 
		cycling regime, can impact assay performance beyond the 
		ability of the current generation of primer design programs to 
		predict. </p>
		<p><b>Primer Tm is key</b></p>
		<p>Maybe not surprisingly, of all primer-specific parameters, empirical 
		testing has indicated that it is the melting temperature (Tm) has the greatest 
		impact on assay performance. Unfortunately, this issue is made more 
		complex by the fact that primer Tm is dependent on the enzyme 
		formulation, a situation exacerbated by the fact that most companies 
		will not disclose the composition of their enzyme formulations. This in 
		turn presents the challenge as to how to effectively predict primer Tm.&nbsp; </p>
		<p>The approach described here originated from the presumption that the 
		reproducibility of Tm prediction is more important than accurately 
		predicting the actual Tm. If true, this would allow large numbers of 
		primers to be designed with confidence that all will have similar Tm&#39;s. 
		As described in another section, the annealing and elongation (A&amp;E) 
		temperature could then be adjusted to match the melting characteristics 
		of the enzyme formulation. </p>
		<p>This then raises the question at to what Tm to target. Although, for 
		the reasons outlined above, the initial choice during early LRE 
		development was somewhat arbitrary in 
		nature, it was surmised that maximizing enzymatic activity could be 
		achieved by selecting a high A&amp;E temperature (i.e. 65-70 oC). However, 
		it was also necessary to limit primer length (i.e. &lt;35 bp), so a 
		compromise Tm of 70 oC was chosen. It was subsequently found that for 
		QuantiTect (the primary enzyme formulation used during develop and testing 
		of LRE) an A&amp;E of 65 oC was optimal. Note however, that A&amp;E temperatures 
		as low as 60 oC have been found to generate no discernable loss in assay 
		performance. </p>
		<p><b>Tm determination via calibration</b></p>
		<p>Particularly in view of the large number of computer programs 
		available for primer design, it was of interest to devise a simple 
		method for designing primers that would be applicable to any 
		primer design program. Indeed, although the method described below is based on IDT&#39;s 
		Oligo Analyzer web interface, the approach has been found to be 
		effective with many other primer design programs...</p>
		<p>&nbsp;www.idtdna.com/analyzer/Applications/OligoAnalyzer</p>
		<p>CAL1 F1: <span style="font-size: 12.0pt; font-family: Arial">
		AGACGAATGCCAGGTCATCTGAAACAG</span><br>
		CAL1 R1: CTTTTGCTCTGCGATGCTGATACCG<br>
		CAL2 F1: GTATCCATCGGGTGTGCTTCCTGATATG<br>
		CAL2 R1: GTGGGTGTGCGACTTAATTCCATCCT</p>
		<p>Enter the CAL1 F1 sequence and press &quot;Analyze&quot;. Using 
		default settings this will generate a predicted Tm of 60.6 oC:</p>
		<p>&nbsp;</p>
		<p align="center"><b>CAL F1: Default settings</b><br>
		<img border="0" src="images/default_tm_determination.gif" width="614" height="151"><br>
		<img border="0" src="images/default_tm_determination2.gif" width="149" height="48"></p>
		<p>In order to &quot;calibrate&quot; the program, the Mg++ concentration 
		can be 
		progressively increased until the predicted Tm increases to 70 oC, which 
		in this example, was 50 mM, generating 
		a predicted Tm of 70.8 oC:</p>
		<p align="center"><b>CAL1 F1: Mg++ Conc adjusted to 50 mM</b><br>
		<img border="0" src="images/adjusted_tm_determination.gif" width="621" height="148"><br>
		<img border="0" src="images/adjusted_tm_determination2.gif" width="147" height="50"></p>
		<p align="left">Note that the choice to increase Mg++ concentration is 
		completely arbitrary and that a predicted Tm only needs to be above 70 
		oC. </p>
		<p align="left">To test the repeatability of the analysis, enter 
		the CAL1 R1 primer sequence and analyze using the same settings:</p>
		<p align="center"><b>CAL1 R1-70.0 oC</b><br>
		<img border="0" src="images/adjusted_cal1r1_tm_determination2.gif" width="151" height="49"></p>
		<p align="left">This generates a predicted Tm of 70.0 oC, indicating 
		that the program can reliably generate an effective primer Tm. This can be 
		tested further by repeated with CAL2 F1 and R1 primers:</p>
		<p align="center"><b>CAL2 F1-70.2 oC</b><br>
		<img border="0" src="images/adjusted_cal2f1_tm_determination2.gif" width="147" height="50"></p>
		<p align="center"><b>CAL2 R1-70.9 oC</b><br>
		<img border="0" src="images/adjusted_cal2r1_tm_determination2.gif" width="151" height="49"></p>
		<p align="left">Thus, for all four primers, the predicted Tm&#39;s fall within the 
		range of&nbsp; 70-71 oC, confirming that this program can generate 
		acceptable reproducibility. Now 
		that the program has been calibrated, new primers can be selected based 
		on a target Tm of 70 oC.</p>
		<p align="left">This can be accomplished by entering a primer sequence 
		of a length that generates a predicted Tm of &gt;70oC, and then to 
		progressively reduce the length until the predicted Tm is close to, but 
		not below 70 oC. For most primers, the length falls in the ~23-33 bp 
		size, depending on the GC content. </p>
		<p align="left">&nbsp;</p>
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